We are studying the two alternatively spliced isoforms of nonmuscle myosin heavy chain II-B, one with an insert of 10 amino acids in loop1 near the ATP-binding domain (B1) and the second with an insert of 21 amino acids in loop2 near the actin-binding domain (B2). Whereas the B1 insert is expressed in neuronal cells early in embryonic development, the B2 insert appears only after birth. In an effort to understand the function of these inserts, the regional expression pattern of each insert was screened employing RT-PCR. The B1 insert was expressed most abundantly in mouse cerebrum, while B2 was enriched in the cerebellum. In situ hybridization revealed that both the B1 and B2 inserts were expressed in cerebellar granule cells. Both the B1 and B2 inserts were also detected in cultured rat granule cells. The expression level of the B2 insert increased with the maturation of granule cells. In contrast, expression of the B1 insert decreased slightly in these cells with maturation. To study the role of these inserts in vivo, we are generating two separate lines of mice in which the exons encoding the B1 and B2 inserts have been replaced by the gene encoding Neomycin resistance (Neo) flanked by LoxP sites, which will allow us to remove Neo, minimizing its possible effect on the phenotype. We are presently assessing these mice for germline transmission. These in situ and in vivo studies should provide useful information on the function of these inserted isoforms.